The Anti-Acne Potential and Chemical Composition of Two Cultivated Cotoneaster Species

In light of current knowledge on the role of reactive oxygen species and other oxidants in skin diseases, it is clear that oxidative stress facilitates inflammation and is an important factor involved in skin diseases, i.e., acne. Taking into consideration the fact that some Cotoneaster plants are valuable curatives in skin diseases in traditional Asian medicine, we assumed that thus far untested species C. hsingshangensis and C. hissaricus may be a source of substances used in skin diseases. The aim of this study was to evaluate the antioxidant, anti-inflammatory, antimicrobial, and cytotoxic activities of their various extracts. LC-MS analysis revealed the presence of 47 compounds (flavonoids, phenolic acids, coumarins, sphingolipids, carbohydrates), while GC-MS procedure allowed for the identification of 42 constituents (sugar derivatives, phytosterols, fatty acids, and their esters). The diethyl ether fraction of C. hsingshangensis (CHs-2) exhibited great ability to scavenge free radicals and good capacity to inhibit cyclooxygenase-1, cyclooxygenase-2, lipoxygenase, and hyaluronidase. Moreover, it had the most promising power against microaerobic Gram-positive strains, and importantly, it was non-toxic toward normal skin fibroblasts. Taking into account the value of the calculated therapeutic index (>10), it is worth noting that CHs-2 can be subjected to in vivo study and constitutes a promising anti-acne agent.


Introduction
The results obtained by Sarici et al. [1] concerning the evaluation of parameters associated with oxidative stress, such as nitric oxide (NO), xanthine oxidase (XO), malondialdehyde (MDA), superoxide dismutase (SOD), and catalase (CAT), in the venous blood of patients using spectrophotometrical methods indicate that oxidative damage of tissues is a significant etiological factor of acne. In view of the above, the role of oxidative stress in the etiology of acne seems to be unquestionable.
The cutaneous propionibacteria (P. acnes, P. avidum, P. granulosum, P. propionicum, and P. lymphophilum) are involved in the maintenance of healthy skin; however, they can also reveal adverse activity as opportunistic pathogens [2]. The three predominant genera, including Propionibacteria, Staphylococci, and Corynebacteria, form the microbial community established on the skin [3].
According to [4], the classification of Propionibacterium is extremely complicated. The major types of Propionibacterium (I, II, III clades) should be classified as P. acnes subsp. acnes, P. acnes subsp. defendens, and P. acnes subsp. elongatum, respectively. The latest research justifies the necessity to divide the genus Propionibacterium into four genera. Additionally, and Iran as therapeutic agents in the treatment of cuts and wounds [13]. In addition, an anti-itching effect of Cotoneaster has been observed [14,15]. According to Akbar [16], Cotoneaster removes hyperpigmentation of the skin.
Taking into consideration the fact that some representatives of Cotoneaster are valuable curatives applied in various skin diseases in traditional Asian medicine, we assumed that thus far untested species-C. hsingshangensis and C. hissaricus-may be a source of active substances used in skin diseases. In view of the above, the present study reports primarily on a thorough examination of the composition (especially phenolic components) and biological activities with an emphasis on dermatological diseases, such as acne. An exploration of Cotoneaster species has been undertaken to provide an explanation for their capability to ameliorate skin conditions.

Preparation of the Extracts
The plant materials were dried in the shade at 24 °C (±0.5 °C) to achieve a constant weight [17]. Extracts were prepared using a mixture of methanol-acetone-water (3:1:1, v/v/v; 3 × 100 mL), and then sonicated at a controlled temperature (40 ± 2 °C) for 30 min [13]. The combined extracts were filtered, concentrated under reduced pressure, and after freezing, lyophilized in a vacuum concentrator (Free Zone 1 apparatus; Labconco, Kansas City, KS, USA) to obtain dried residues. The obtained extracts were dissolved in hot water,

Preparation of the Extracts
The plant materials were dried in the shade at 24 °C (±0.5 °C) to achieve a constant weight [17]. Extracts were prepared using a mixture of methanol-acetone-water (3:1:1, v/v/v; 3 × 100 mL), and then sonicated at a controlled temperature (40 ± 2 °C) for 30 min [13]. The combined extracts were filtered, concentrated under reduced pressure, and after freezing, lyophilized in a vacuum concentrator (Free Zone 1 apparatus; Labconco, Kansas City, KS, USA) to obtain dried residues. The obtained extracts were dissolved in hot water,

Preparation of the Extracts
The plant materials were dried in the shade at 24 • C (±0.5 • C) to achieve a constant weight [17]. Extracts were prepared using a mixture of methanol-acetone-water (3:1:1, v/v/v; 3 × 100 mL), and then sonicated at a controlled temperature (40 ± 2 • C) for 30 min [13]. The combined extracts were filtered, concentrated under reduced pressure, and after freezing, lyophilized in a vacuum concentrator (Free Zone 1 apparatus; Labconco, Kansas City, KS, USA) to obtain dried residues. The obtained extracts were dissolved in hot water, filtered after 24 h, and subjected to liquid-liquid extraction with diethyl ether, ethyl acetate, and n-butanol, successively. The obtained fractions of diethyl ether, ethyl acetate, and n-butanol, as well as the water residue, were evaporated in vacuo and lyophilized using a vacuum concentrator.

Total Flavonoid, Phenolic, and Phenolic Acids Content
Total flavonoid (TFC) and total phenolic content (TPC) were established using colorimetric assays as described previously [18]. The absorbance was measured at 430 and 680 nm, respectively, using a Pro 200F Elisa Reader (Tecan Group Ltd., Männedorf, Switzerland). TPC was estimated from the calibration curve (R 2 = 0.9845), using gallic acid as a standard (concentration ranged 0.002-0.1 mg/mL). The results were expressed as mg of gallic acid equivalent (GAE) per 1 g of dry extract (DE). TFC was estimated from the calibrated curve (R 2 = 0.995), using quercetin (0.004-0.11 mg/mL) as a standard. The results were expressed as mg of quercetin equivalent (QE) per 1 g of DE. Total phenolic acid (TPAC) content was assessed using Arnov's reagent as described in the Polish Pharmacopoeia IX (an official translation of PhEur 7.0) [17]. The absorbance was measured at 490 nm. TPAC was estimated from the calibration curve (R 2 = 0.9999), using caffeic acid as a standard in a concentration of 3.36-23.52 µg/mL. The results were ex-pressed as mg of caffeic acid equivalent (CAE) per 1 g of DE.

LC-MS Analysis
The chromatographic measurements were performed using the LC/MS system from Thermo Scientific (Q-EXATCTIVE and ULTIMATE 3000, San Jose, CA, USA) equipped with an ESI source. The ESI was operated in negative polarity modes under the following conditions: spray voltage-3.5 kV; sheath gas-40 arb. units; auxiliary gas-10 arb. units; sweep gas-10 arb. units; and capillary temperature-320 • C. Nitrogen (>99.98%) was employed as sheath, auxiliary, and sweep gas. The scan cycle used a full-scan event at a resolution of 60,000. A Gemini C18 column (4.6 × 100 mm, 3 µm) (Phenomenex, Torrance, CA, USA) was employed for chromatographic separation, which was performed using gradient elution. Mobile phase A was 25 mM formic acid in water; mobile phase B was 25 mM formic acid in acetonitrile. The gradient program started at 5% B, increasing to 95% for 60 min, followed by isocratic elution (95% B) for 10 min. The total run time was 70 min at the mobile phase flow rate 0.4 mL/min. The column temperature was 25 • C. In the course of each run, MS spectra in the range of 100-700 m/z were collected continuously. Additionally, the MS2 functions were used to carry out a detailed qualitative analysis. The collision energy for each examined compound was 25%.
The amounts of the identified compounds were carried out based on the calibration curves obtained for the standard. In the case of quantitative analysis of compounds that do not have standards, calibration curves for substances of similar structure were used. All the results are presented as the mean of three independent measurements (n = 3).

GC-MS Analysis
The qualification of the sample extract was performed using a GC-MS/MS system (GCMS-TQ8040; Shimadzu, Kyoto, Japan) equipped with a ZB5-MSi fused-silica capillary column (30 m × 0.25 mm i.d., 0.25 µm film thickness; Phenomenex, Torrance, CA, USA). Grade 5.0 helium was used as the carrier gas. Column flow was 1 mL/min. The injection of a 1 µL sample was performed using an AOC-20i + s type autosampler (Shimadzu, Kyoto, Japan). The injector was working at a temperature of 310 • C. The following temperature program was applied: the oven temperature was held at 60 • C for 2 min and was subsequently increased linearly at a rate of 6 • C/min to 310 • C, where it was held for 15 min. The mass spectrometer was operated in EI mode at 70 eV; the ion source temperature was 225 • C. The mass spectra were measured in the range 40-450 amu. The amounts of the individual analytes were estimated by the peak normalization method.

Antioxidant Activity
All antioxidant and enzyme inhibitory assays were done in 96-well plates (Nunclon, Nunc, Roskilde, Denmark) using Infinite Pro 200F Elisa Reader (Tecan Group Ltd., Männedorf, Switzerland). The experiments were performed in triplicate.

DPPH • Assay
The 2,2-diphenyl-1-picryl-hydrazyl (DPPH • ) free radical scavenging activity of Cotoneaster extracts and the positive control-ascorbic acid (AA)-was studied using the method described previously [18], but with some modifications. After 30 min of incubation at 28 • C, the decrease in DPPH • absorbance, caused by the tested extracts, was measured at 517 nm. The results were expressed as values of IC 50 .

ABTS •+ Assay
The ABTS •+ decolorization assay was the second method applied for the assessment of antioxidant activity [18]. The absorbance was measured at 734 nm. Trolox was used as a positive control. The results were expressed as values of IC 50 .

Metal Chelating Activity (CHEL)
The metal chelating activity was established using the method described by Guo et al. [19], modified in our previous study [18,20]. The absorbance was measured at 562 nm. As a positive control, Na 2 EDTA*2H 2 O was used. Results were expressed as the IC 50 values of the Cotoneaster extracts based on concentration-inhibition curves.
2.8. Enzyme Inhibitory Activity 2.8.1. Cyclooxygenase-1 (COX-1) and Cyclooxygenase-2 (COX-2) Inhibitory Activity The extracts of Cotoneaster species were examined for cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) inhibitory activity using a COX (ovine/human) Inhibitor Screening Assay Kit (Cayman Chemical, MI, USA) according to the protocol of the manufacturer. The extracts were tested at different concentrations. Indomethacin was used as a positive control.

Lipoxygenase Inhibitory Activity
Anti-lipoxygenase activity of Cotoneaster extracts was determined using the Lipoxygenase Inhibitor Screening Assay Kit (Cayman Chemical, MI, USA) according to the protocol of the manufacturer. The extracts were tested at different concentrations. The effective concentration (µg/mL) in which lipoxygenase activity is inhibited by 50% (IC 50 ) was estimated graphically. Nordihydroguaiaretic acid (NDGA) was used as a positive control.

Hyaluronidase Inhibitory Activity
Anti-hyaluronidase activity was established using the method described by Liyanaarachchi et al. [21]. After 20 min incubation at 37 • C, the absorbance was measured at 585 nm. The extracts were tested at different concentrations. Epigallocatechin gallate was used as a positive control.

Bacterial Strains
The antibacterial power of Cotoneaster extracts was determined using bacterial strains causing skin diseases (including seborrhea and acne). We used microaerobic Gram-positive bacteria: Cutibacterium granulosum PCM 2462, C. acnes PCM 2334, C. acnes PCM 2400, (possessed from the Polish Collection of Microorganisms PCM Institute of Immunology and Experimental Therapy Polish Academy of Sciences, Poland, as Propionibacterium, now-Cutibacterium), C. acnes ATCC 11827; aerobic Gram-positive: Staphylococcus epidermidis ATCC 12228 and S. aureus ATCC 25923; and aerobic Gram-negative strains Pseudomonas aeruginosa ATCC 27853 and Escherichia coli ATCC 25992. Each bacterial strain was preincubated overnight at 37 • C on agar plates. Mueller-Hinton (BioMaxima S.A., Lublin, Poland) agar or broth (MH-agar, MH-broth) for aerobic strains and Brain-Heart Infusion (Oxoid Ltd, Altrincham, England) agar or broth (BHI-agar, BHI-broth) for microaerobic bacteria were used. The bacteria were suspended in 5 mL of sterile saline water, and the absorbance of this inoculum was adjusted to 108 CFU/mL (0.5 Mc'Farland scale).

Disc Diffusion Assay
This disc diffusion assay can evaluate the antibacterial potency of tested extracts and was made according to proven methods [22,23]. Briefly, solid medium in Petri dishes was smeared with inoculum of 0.5 Mc'Farland. The plant extracts were dissolved in DMSO (10 mg/mL) then loaded over sterile filter paper discs (8 mm in diameter) to obtain a final concentration of 0.1 mg per disc. Inoculated plates with samples were incubated at 37 • C for 24 h (aerobic stains) or 48 h (microaerobic bacteria).
The zones of growth inhibition around plant samples were measured [mm] and recognized as antibacterial potency. The larger the zone of growth inhibition, the greater the antibiotic activity.

Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) Determination
The MIC is the lowest concentration of a test compound that inhibits microbial proliferation. The test examined the MIC of the Cotoneaster extracts against Staphylococcus aureus ATCC 25923, S. epidermidis ATCC 12228, C. acnes ATCC 11827, C. acnes PCM 2334, C. acnes PCM 2400, and C. granulosum PCM 2462, whose optical density was 0.5 Mc'Farland scale. Double microdilution in the 96-well plate assay was used according to the CLSI method with some modification, as described in our paper [23,24]. Such an amount of the extract solution to the wells was added, that using the principle of double microdilution to obtain final concentrations in the range of 3.9-1000 µg/mL. Then, each well was inoculated (2 µL of inoculum density of 0.5 Mc'Farland). A background control (broth with extract), a negative control (the broth alone), and a positive control (broth and inoculum) were also prepared. The plates were incubated under conditions proper to bacterial growth (aerobic bacteria at 37 • C, for 24 h; anaerobic bacteria at 37 • C, for 48 h). Finally, microbial growth was determined using a BioTek Synergy H4 (BioTek, Winooski, VT, USA) plate reader at a wavelength of 600 nm.
MBC is the lowest concentration of the agent with bactericidal properties. To determine this, 96-well plates obtained in the previous experiment were used to determine the MIC of the tested extracts. Ten µL of solutions taken from the wells in which no bacterial growth was observed were applied to the new Petri plates. Then, the plates were incubated under optimal conditions for the growth of the given bacteria. After the incubation was completed, a visual assessment of the agar surface of the plates was made. The MBC was considered to be the one in which there was no visible bacterial growth on the solid medium.
To visualize the obtained results, it is advantageous to present the MBC/MIC ratio. Thus, MBC/MIC values ≤4 demonstrate that the agent is bactericidal. MBC/MIC values >4 indicate the bacteriostatic nature of the tested agents [25].
Microbiological tests were performed in three separate experiments (n = 3).

Cytotoxic Activity
This experiment was carried out according to the protocol described by us previously [26]. The cytotoxic activity of selected substances was evaluated toward the BJ cell line (normal human fibroblasts, ATCC CRL-2522TM). Briefly, the BJ cells were seeded in 96well plates, and then after 24 h incubation at 37 • C in suitable conditions (5% CO 2 , 95% air), the culture medium was replaced with two-fold serial dilutions of investigated substances (1.95-1000 µg/mL). After 24 h incubation, the cell viability was assessed using the MTT assay. The obtained data were presented as mean values ± standard deviations (SD). These results were subjected to four-parameter nonlinear regression analyses (GraphPad Prism 5,

Statistical Analysis
The results were expressed as mean values ± standard deviation (SD) of three independent experiments. The data from cell culture experiments were subjected to statistical analysis using unpaired Student's t-test or One-Way ANOVA test, followed by a Tukey's multiple comparison test, and differences were considered significant when p < 0.05 (Graph-Pad Prism 5, version 5.04, GraphPad Software, San Diego, CA, USA), whereas Principal Component Analysis was carried out in R version 3.6.3 (64-bit, Windows 10), using built-in "prcomp" function.

Phytochemical Analysis
Total phenolic content (TPC) for Cotoneaster extracts and fractions was determined using Folin-Ciocalteu reagent, and the results were estimated as gallic acid equivalents (GAE) per g of dry extract (DE) ( Table 1). Our results showed that the leaves of C. hissaricus (CHi) have the highest phenolic content (296.13 ± 1.52 mg GAE/g DE) than C. hsingshangensis (CHs) (193.84 ± 1.14 mg GAE/g DE). The results obtained in our study were better compared to data presented for extracts derived from the leaves of other Cotoneaster species. For instance, Kicel et al. [27] demonstrated that TPC for the 70% aqueous methanolic extracts of leaves of different Cotoneaster species cultivated in Poland varied from 51.7 (C. tomentosus) to 154.3 mg GAE/g (C. bullatus) of dry weight of the plant material. The same authors [28] also found lower amounts of phenolic compounds in 70% aqueous methanolic extracts of the fruits (from 26 to 43.5 mg GAE/g PM) of different Cotoneaster species. CHi-methanol-acetone-water (3:1:1, v/v) extract of C. hissaricus; CHs-methanol-acetone-water (3:1:1, v/v) extract of C. hsingshangensis; CHi-1-water fraction of C. hissaricus, CHi-2-diethyl ether fraction of C. hissaricus, CHi-3-butanol fraction of C. hissaricus, CHi-4-ethyl acetate fraction of C. hissaricus; CHs-1-water fraction of C. hsingshangensis, CHs-2-diethyl ether fraction of C. hsingshangensis, CHs-3-butanol fraction of C. hsingshangensis, CHs-4-ethyl acetate fraction of C. hsingshangensis. Values were presented as mean ± standard deviation (n = 9). Statistical analysis: a-significantly different results compared to CHi; b-significantly different results compared to CHi-1; c-significantly different results compared to CHi-2; d-significantly different results compared to CHi-3; e-significantly different results compared to CHs; f-significantly different results compared to CHs-1; g-significantly different results compared to CHs-2; h-significantly different results compared to CHs-3; i-significantly different results compared to CHs-4; One-Way ANOVA test, followed by a Tukey's multiple comparison test, p < 0.05.
In our study, we also examined the content of phenolics in fractions obtained after fractionating crude extracts between solvents of different polarity. The results showed that the TPC level was in the range of 83.87 ± 0.23 (water fraction of C. hissaricus) to 559.77 ± 3.76 mg GAE/g DE (ethyl acetate fraction of C. hsingshangensis). Kicel and co-authors [29] obtained higher values with the highest TPC level for ethyl acetate fractions (470.9-650.8 mg GAE/g dw) and diethyl-ether fractions (453.1-546.9 mg GAE/g dw) of defatted methanol extracts.
Thus, the results obtained in our study indicated that the leaves of C. hissaricus and C. hsingshangensis are a rich source of phenolic compounds.
The total flavonoid content of the leaves of C. hissaricus and C. hsingshangensis was estimated by a previously described colorimetric method [27]. The data were expressed as quercetin equivalents (QE) per g of dry extract (DE). The results presented in Table 1 show that the content of total flavonoids in both species is at an average level. The comparable content was noted for the leaves of C. hissaricus (CHi) and C. hsingshangensis (CHs) (25.38 ± 2.35 and 47.72 ± 0.37 mg QE/g DE, respectively). The results obtained in our study were higher than those found by Mahmutović-Dizdarević et al. [11]. In their study, quantitative estimation revealed that methanolic extracts from leaves of C. tomentosus possessed 18.17 ± 0.30 mg QE/g dw. flavonoid content, followed by C. integerrimus-16.42 ± 0.35 mg QE/g dw. and C. horizontalis-10.55 ± 0.51 mg QE/g dw. Moreover, they found that fruits of these three species contain a lower amount of flavonoids-2.76-9.38 mg QE/g dw. A higher amount of flavonoids was found in the methanolic extract of leaves of C. wilsonii Nakai (36.46 ± 1.89 mg QE/g dw), while in the stems and fruits, the content of flavonoids was lower (6.09 ± 0.71 and 0.23 ± 0.20 mg QE/g dw, respectively) [30]. Kicel et al. [29] also obtained lower results for defatted methanol extracts of the three Cotoneaster species cultivated in Poland, and the values were from 14.70 ± 0.05 to 67.71 ± 0.80 mg/g dw. These authors obtained slightly higher results for the fractions obtained from crude extracts compared to the results in our study. The highest level of flavonoids was determined for the ethyl acetate fraction of C. integerrimus leaves (403.56 ± 7.48 mg/g dw). In our study, the lowest amount of total flavonoids was found in water fractions of C. hissaricus and C. hsingshangensis (0.12 ± 0.05 and 0.23 ± 0.02 mg QE/g DE, respectively) and the highest was in the ethyl acetate fraction of C. hsingshangensis (252.27 ± 0.24 mg QE/g DE).
The total phenolic acid content (TPAC) in Cotoneaster extracts is presented in Table 1. As for the content of polyphenols and flavonoids, a higher content of phenolic acids was noted for crude extract (61.27 ± 0.93 mg CAE/g DE) and fractions (19.65 ± 0.30-91.95 ± 0.48 mg CAE/g DE) of C. hsingshangensis.
The use of plants in the cure of different diseases depends on their phytochemical composition. Our research of the presence of active compounds in the crude methanolacetone-water (3:1:1, v/v/v) extracts and different fractions (diethyl ether, ethyl acetate, butanol, and aqueous residual) indicated that methanol-acetone-water extracted the greatest range of constituents from the leaves of both Cotoneaster species. Therefore, in our study, we used only the crude extracts for the LC-MS and GC-MS analyses.
Thus, in the next step of our study, the chemical composition of the extracts obtained from Cotoneaster extracts was investigated using the LC-MS method. Table 2 shows 47 identified compounds, including their molecular formula, theoretical and experimental molecular mass, both errors in ppm and mDa, and the fragments. In our study, flavonoids, phenolic acids, coumarins, cyanogenic glycosides, sphingolipids, and carbohydrates were identified using LC-MS analysis. The chromatograms with marked main peaks are displayed in Figures 3 and 4. The results of the quantitative analysis are presented in Table 3. The amounts of the identified compounds were carried out based on the calibration curves obtained for the standard. In the case of quantitative analysis of compounds that do not have standards, calibration curves for substances of similar structure were used.          Among flavonoids, the most abundant in both species were quercetin derivatives. In the leaves of C. hissaricus rutin (18,028 ± 650 µg/g DE), isoquercitrin (10,079 ± 353 µg/g DE), hyperoside (9119 ± 331 µg/g DE), 5,7,2 ,5 -tetrahydroxyflavanone 7-O-glucoside (8067 ± 290 µg/g DE), and quercetin 3-O-(2 -O-xylosyl)galactoside (7318 ± 289 µg/g DE) were found in the largest amount. In the leaves of C. hsingshangensis, isoquercitrin (8926 ± 327 µg/g DE), quercitrin (6726 ± 251 µg/g DE), and hyperoside (6184 ± 237 µg/g DE) were observed in the greatest amount. Isoquercitrin, rutin, hyperoside, and quercitrin were previously identified as dominant in the other Cotoneaster species [27,28,[31][32][33][34][35][36][37]. Vitexin 2 -O-arabinoside, vitexin-2 -O-rhamnoside, and 5-methylgenistein, which were found in large quantities in both studied species, were previously noticed only in leaves of C. thymaefolia [32], aqueous alcoholic extracts of leafy twigs of C. obricularis [38], and chloroform extracts of C. simonsii leafy twigs [39], respectively. Subsequent compounds of rare occurrence in the Cotoneaster genus are 5,7,2 ,5 -tetrahydroxyflavanone and its 7-O-glucoside, which were identified in C. thymaefolia leaves [32]. It is worth noting that rare in nature flavonoids, such as biochanin A 7-O-glucoside (sissotrin) and 5-methylgenistein-4 -O-glucoside were observed in both studied species. Previously, sissotrin was found only in the n-butanol fraction of the methanolic extract of leaves of C. mongolica [37], methanol extract of C. serotina flowers [40], and methanol extract of flowers and fruits of C. pannosa [40]. 5-methylgenistein-4 -O-glucoside was previously observed only in the chloroform extract of leafy twigs of C. simonsii [39]. Quercetin 3-O-gentiobioside (quercetin 3-O-β-D-glucopyranosyl(l-6)glucopyranoside) is noteworthy regarding the fact that it was one of the first compounds isolated from the Cotoneaster genus and is infrequent, being only reported in C. oligantha stems [41]. Furthermore, kaempferol 3-O-glucoside (astragalin) is of rare occurrence in the Cotoneaster genus and reported only in the methanol extract of C. mongolica leaves by Odontuya and co-authors [37].
The second largest group of active compounds were phenolic acids, and among them, chlorogenic acid, gentisic acid 2-O-glucoside, and caffeoylmalic acid were the most abundant in both species. Apart from these typical phenolic acids, cotonoate A and horizontoate A were observed in the leaves of C. hissaricus and C. hsingshangensis, but only in C. hissaricus was cotonoate A identified in quantifiable amounts (1564 ± 55 µg/g DE).
In previous research, cotonoate A and horizontoate A were isolated only from the leafy twigs of the C. racemiflora chloroform soluble fraction of the methanolic extract [14] and methanolic extract of C. horizontalis [15], respectively.
Chlorogenic acid seems to be a ubiquitous ingredient among the Cotoneaster genus. What most attracts attention is that both studied extracts of leaves of C. hissaricus and C. hsingshangensis constitute a significant percentage of this acid (37,932 ± 1330 and 60,043 ± 2231 µg/g DE, respectively). According to [42], this compound participates in diminishing P. acnes-induced matrix metalloproteinase-9 levels, obstructing nuclear factor-κB (NF-κB) activation, inactivating mitogen-activated protein kinases (MAPK), and decreasing the migration of neutrophils and interleukin (IL)-1β+ populations in vivo. Therefore, it is increasingly being recognized as possibly efficacious in the management of dermatological conditions, such as acne.
Among the other polyphenolic compounds, some coumarins were identified in the leaves of C. hissaricus and C. hsingshangensis, and the most abundant in both species was scopoletin (12,219 ± 440 and 10,481 ± 371 µg/g DE, respectively). This compound was isolated earlier from the leafy twigs of C. racemiflora from methanolic extract [43] and the ethyl acetate-soluble fraction of methanolic extract [44,45].
In both studied species, great amounts of mannitol were also observed (C. hissaricus-6834 ± 249 µg/g DE and 3104 ± 123 µg/g DE-C. hsingshangensis). This compound is the predominant polysaccharide of manna, which is produced by the young shoots of C. discolor, C. nummularius, C. tricolor, and C. nummularioides [46]. From cyanogenic glycosides, prunasin, and amygdalin were found. These glycosides were also previously identified in the fruits and leaves of C. congesta, C. praecox, and C. integerrimus [47] and in the ethanol extract of C. horizontalis leafy twigs [48].
The appropriate GC-MS procedure allowed for the identification of 42 and 41 compounds in the leaves of C. hissaricus (Table 4, Figure 5) and C. hsingshangensis (Table 5, Figure 6), respectively. Among the identified compounds, four main groups of analytes can be distinguished-sugar derivatives, phytosterols, fatty acids, and their esters.        The dominant group of compounds included in the extract from C. hissaricus are sugar derivatives; their content exceeds 37%. 1,3:2,5-dimethylene-l-rhamnitol, the content of which in the extract is 12.29%, and 2-deoxy-D-galactose, the content of which is 14.62%, are noteworthy. In the case of the extract of C. hsingshangensis, the content of sugar derivatives does not exceed 30%. The estimated amount of 1,3:2,5-dimethylene-L-rhamnitol (14.62%) is at a similar level of concentrations, while the content of 2-deoxy-D-galactose is nearly three times lower. The second major group of identified compounds is phytosterols. The content of those compounds in the extract of C. hissaricus exceeds 21%, while the amount of the main phytosterol, phytol acetate, is 15.79%. The extract of C. hsingshangensis, in turn, contains an approximately 25% higher concentration of phytosterols (more than 26%). The third major group of compounds in both extracts was fatty acids and their esters. Among the most important are hexa-decanoic acid, methyl ester (which constitutes 10.07%-CHi; 17.02%-CHs), followed by palmitic acid (6.09%-CHi; 5.14%-CHs), and linolenic acid (2.14%-CHi; 3.39%-CHs). Linolenic and palmitic acids were previously recorded as the major fatty acids in the fruits of C. zabelii, C. splendens, C. hjelmqvistii, and C. horizontalis [28] and the aerial parts of C. horizontalis [55].
It Is worth mentioning that the pronounced bioactivity of phenols toward skin disorders is enhanced by the simultaneous presence of such components as 6-hydroxy-4,4,7atrimethyl-5,6,7,7a tetrahydrobenzofuran-2(4H)-one, which was reported as a strong antiinflammatory agent [56], and which was identified in both studied Cotoneaster species.
A broad variety of active compounds in Cotoneaster species lead to overlapping activities of miscellaneous agents, hence acting on several levels of different diseases caused by oxidative stress is possible.

Biological Activity
Taking into account that both oxidation and inflammation play significant roles in disease pathogenesis, recent research has focused on the evaluation of the antioxidant and anti-inflammatory activity of plants. In our study, we examined in vitro antioxidant, anti-cyclooxygenase, anti-lipoxygenase, anti-hyaluronidase, antibacterial, and cytotoxic activities of leaves of C. hissaricus and C. hsingshangensis.
Phenolic compounds can reduce oxidative stress by several mechanisms that depend on their chemical structure. One of them is the chelation of metal ions, such as iron, which plays a key role in the production of damaging oxygen species [58].
The chelating ability was determined based on measurement of the percentage of inhibition of formation of the ferrozine-Fe 2+ complex. As reported in Table 6, the extracts and fractions from leaves of both studied Cotoneaster species possessed the capacity to interfere with the formation of iron and ferrozine complexes, which suggests their high chelating capacity and ability to capture iron ions before ferrozine. The IC 50 values of most of these extracts showed higher chelating activity than the positive control-Na 2 EDTA*2H 2 O (IC 50 = 4.15 ± 0.10 µg/mL). Among the investigated extracts, the best activity was noticed in the ethyl acetate fraction of C. hsingshangensis (IC 50 = 0.50 µg/mL). Zengin and coauthors [57] also evaluated the metal chelating capacity of different extracts of aerial parts of C. nummularia. They found that the highest chelating activity was expressed by the ethyl acetate extract (IC 50 = 0.25 mg EDTA/g of extract). Among the ethanol extracts of 34 species belonging to Rosaceae, Cotoneaster meyeri, C. morulus, and C. numullaria caused moderate metal-chelating effects (5.91, 21.48, and 26.19 chelation%, respectively) [34].

Enzyme Inhibitory Activity
Lipoxygenases (LOXs) are present in the human body and play a crucial role in the stimulation of inflammatory reactions. Exaggerated quantities of reactive oxygen species can induce inflammation that stimulates the release of cytokines and then the activation of lipoxygenases. They are connected with the spread of many diseases, and their inhibition is viewed as a relevant step in their prevention [59]. The lipoxygenase inhibitor screening assay kit is a popular method for lipoxygenase detection, which estimates the presence of hydroperoxides (4-hydroperoxy cis-trans 1,3-conjugated pentadienyl moiety within the unsaturated fatty acid) at various positions (5, 12-, 15-), which are produced in the lipoxygenation reaction using a purified lipoxygenase.
Sinha et al. [60] noted that perioxisome proliferator activated receptor (PRAR) ligands induce lipogenesis in cultured human sebocytes; hence, 5-lipoxygenase inhibitors demonstrate the ability to decline acne lesions, due to the fact that they possess the capability to reduce lipogenesis. The presence of PPARα receptors within sebocytes has been observed in peroxisomes, mitochondria, and microsomes.
The results of the inhibition of lipoxygenase are shown in Table 7. The diethyl ether fraction (CHs-2) and ethyl acetate fraction (CHs-4) of C. hsingshangensis showed a considerable ability to inhibit lipoxygenase activity (IC 50 = 4.15, and 5.72 µg/mL, respectively), while the water fraction of C. hissaricus showed the lowest activity (IC 50 = 129.46 µg/mL). Both CHs-2 and CHs-4 exhibited significantly higher inhibitory activity than that of nordihydroguaiaretic acid (NDGA) used as a positive standard (IC 50 = 5.89 µg/mL). In a recent study of Cotoneaster species in regard to their capacity to inhibit lipoxygenase, it was found that different extracts of the leaves of C. bullatus, C. integerrimus, and C. zabelii have moderate activity with IC 50 values in the range of 95.19 to 684.84 µg/mL. The best LOX inhibition was achieved by the butanol fraction of C. bullatus (IC 50 = 95.19 µg/mL) [29]. The hydromethanolic extracts of fruits of nine Cotoneaster species cultivated in Poland showed the strongest inhibition of LOX, with IC 50 values in the range of 62.54 (C. zabelii) to 165.76 (C. nanshan) µg/mL [28]. Table 7. Anti-lipoxygenase, anti-hyaluronidase, and anti-cyclooxygenase activities of the leaves of C. hissaricus and C. hsingshangensis. Skin is especially sensitive to reactive oxygen species because it is exposed to oxidative stress from both endogenous and exogenous sources. Although oxidative stress is a key factor in this process, hyaluronic acid also plays a notable role. Its integrity inside the dermal matrix is substantial for cell integrity and proliferation. Under oxidative stress, hyaluronidase, which is responsible for hyaluronic acid depolymerization, is overactivated and breaks down this anionic glycosaminoglycan, carrying on to the destruction of the proteoglycan system. This results in the deregulation of skin homeostasis and increases inflammatory and allergic conditions [61,62].

Sample
In our study, the ethyl acetate fraction of C. hsingshangensis exhibited the best hyaluronidase inhibition activity (IC 50 = 1.89 µg/mL) compared to the other extracts (Table 7). Most importantly, such activity was even better than the positive standard-EGCG (IC 50 = 6.25 µg/mL). In turn, the IC 50 values for the crude extracts of C. hissaricus and C. hsingshangensis were 15.09 ± 0.61, and 6.82 ± 0.15 µg/mL, respectively. Previous studies have shown that methanol extracts of the leaves of C. bullatus, C. integerrimus, and C. zabelii reduced the activity of hyaluronidase in a concentration-dependent manner. The most active were the butanol fraction of C. bullatus and C. zabelii (IC 50 = 2.81, and 6.08 µg/mL, respectively) and they had inhibition activity better than indomethacin used as a positive control (IC 50 = 8.61 µg/mL) [29]. Moreover, methanol extracts of fruits of C. bullatus, C. dielsianus, C. divaricatus, C. hjelmqvistii, C. horizontalis, C. lucidus, C. nanshan, C. splendens, and C. zabelii showed moderate inhibition of LOX with IC 50 values in the range of 25.65 (C. lucidus) to 45.64 µg/mL (C. nanshan) [28].
Cyclooxygenases catalyze two reactions, the first being a cyclooxygenase function consisting of the addition of molecular oxygen to arachidonic acid to form prostaglandin G 2 (PGG 2 ). The second is the conversion of PGG 2 to PGH 2 by a peroxidase function. Therefore, this enzyme performs the initial reaction in the arachidonic acid metabolic cascade, carrying on to the formation of pro-inflammatory, i.e., prostaglandins, which regulate smooth muscle contractility, platelet aggregation, and mediate pain. Cyclooxygenase constitutive (COX-1) is responsible for the maintenance of physiological prostanoid biosynthesis, and COX-2 (an inducible isoform) is connected to inflammatory cell types and tissues [63].

Antibacterial Activity Diffusion Test in Solid Medium
The antibacterial activity of the Cotoneaster extracts and fractions was assessed by diffusion test in a solid medium and measuring the zones of bacterial growth inhibition. The larger the zone around the applied samples, the more active the extract/fraction is.
The data show (Figure 7, Table S1) that the Cotoneaster fractions, not the starting crude extracts, possessed the strongest antimicrobial properties. The largest zones of growth inhibition of Gram-positive microaerobic bacteria were produced by CHs-2 fraction (23 mm-21 mm), then CHs-4 (19 mm-16 mm), CHs-1 (15 mm-14 mm), and CHs-3 fractions (14 mm-11 mm). These fractions against Gram-positive aerobic strains showed weaker activity (in the range 19 mm-6 mm) than against microaerobic bacteria. Crude Cotoneaster extracts CHi and CHs showed moderate activity against all tested Gram-positive strains (12 mm-6 mm). None of the samples were active against Gram-negative bacteria. Previous studies have reported the resistance of both Gram-positive and Gram-negative bacteria to the leaf and bark of C. integerrimus, C. tomentosus, and C. horizontalis methanolic extracts. Mahmutović-Dizdarević and co-authors [11] found that these extracts have significant antimicrobial activity against Salmonella enterica, Pseudomonas aeruginosa, Escherichia coli, Extended Spectrum Beta-Lactamase producing E. coli or ESBL E. coli, Enterococcus faecalis, Staphylococcus aureus, Methicillin-resistant Staphylococcus aureus or MRSA, and Bacillus subtilis. In a further study, the antibacterial activity of the ethanolic extract of roots of C. acuminatus was also investigated against Bacillus pumilus, B. subtilis, E. coli, Microccocus glutamicus, S. aureus, Proteus vulgaris, and P. aeruginosa. The greatest effect was found for 100 µg/mL extract, with growth inhibition zones 10-18 mm [64]. Using the broth microdilution method, the antibacterial activity of the water, methanol, and ethyl acetate extracts of C. nummularia was evaluated against E. coli, Bacillus cereus, P. aeruginosa, methicillin sensitive S. aureus (MSSA), Klebsiella pneumoniae, Salmonella enteritidis, S. pneumoniae, Sarcina lutea, Enterococcus faecalis, methicillin resistant S. aureus (MRSA), and strains of methicillin resistant S. aureus isolated from clinical samples. The authors found that E. faecalis was the most sensitive bacteria, and B. cereus, K. pneumoniae, and S. enteritidis were the most resistant bacteria against all extracts except for the ethyl acetate extract [57]. To the best of our knowledge, this study represents the first report of the antibacterial activity against C. acnes, S. epidermidis, and C. granulosum strains, which can be responsible for skin diseases of Cotoneaster species.

Results of MIC and MBC Determination
In order to determine the MIC values of the selected active fractions of crude C. hsingshangensis extract (CHi, CHs, CHs-1-CHs-4), an assay was performed using the double dilution method in a 96-well plate. After the plates were incubated under the appropriate conditions for the given strain, they were analyzed in an automatic plate reader (at 600 nm) against the control. The concentration at which no bacterial growth was observed was taken as the MIC. Next, the MBC value was determined by dispensing 10 μL of the solutions taken from the wells of 96-well plates in which no bacterial growth was observed.
The data contained in Table 8 confirm the results obtained in the earlier diffusion assay; namely, the tested fractions are the most active against the acne strains of Cutibacterium spp.  To the best of our knowledge, this study represents the first report of the antibacterial activity against C. acnes, S. epidermidis, and C. granulosum strains, which can be responsible for skin diseases of Cotoneaster species.

Results of MIC and MBC Determination
In order to determine the MIC values of the selected active fractions of crude C. hsingshangensis extract (CHi, CHs, CHs-1-CHs-4), an assay was performed using the double dilution method in a 96-well plate. After the plates were incubated under the appropriate conditions for the given strain, they were analyzed in an automatic plate reader (at 600 nm) against the control. The concentration at which no bacterial growth was observed was taken as the MIC. Next, the MBC value was determined by dispensing 10 µL of the solutions taken from the wells of 96-well plates in which no bacterial growth was observed.
Next, the MBC-as the lowest concentration of an antibacterial sample required to kill the bacterium-can be assayed based on an MIC test by subculturing samples on agar plates. Thus, the clear medium from MIC plates was spread on fresh agar to check its sterility. The concentration of the sample that did not produce colonies was considered the MBC power. According to the ratio MBC/MIC, we visualized and appreciated antibacterial activity. If the ratio MBC/MIC ≤ 4, the effect was considered bactericidal, but if the ratio MBC/MIC > 4, the effect was defined as bacteriostatic. Data of MBC/MIC ratio displayed in Table 8 show that none of C. hsingshangensis fractions showed any bactericidal activity in relation to the acne bacteria. The most promising power against microaerobic Grampositive strains was displayed for CHs-2 (diethyl ether fraction of C. hsingshangensis). The remaining extract power (CHs-1, CHs-3, CHs-4) was not measurable.

Cytotoxic Activity
The obtained data revealed that all Cotoneaster extracts possessed low cytotoxicity toward normal human fibroblasts (Figure 8), as it was not possible to determine their CC 50 values at tested concentrations (1.95-1000 µg/mL). The CC 50 value denotes the extract concentration that inhibits BJ viability to 50%. Among the tested Cotoneaster extracts, CHs-3 exhibited the lowest ability to inhibit the viability of BJ cells because at the highest tested concentration (1000 µg/mL), the cell viability was 77.06 ± 1.57%. For comparison, fibroblast viability treated with CHi, CHs, CHs-1, and CHs-4 at the same concentration was 60.60 ± 1.47%, 53.18 ± 1.84%, 68.28 ± 5.55%, and 63.80 ± 4.39%, respectively. In the case of CHs-2, cell viability after incubation at concentrations of 250 µg/mL, 500 µg/mL, and 1000 µg/mL was 54.40 ± 3.56%, 43.35 ± 2.02%, and 45.89 ± 4.57%, respectively. Thus, these results may suggest that the CC 50 value for this substance should be detected in the range 250-500 µg/mL. As a consequence, it was demonstrated that CHs-2 possessed the highest ability to inhibit BJ cell viability compared to other substances.

Multivariate Analysis of the Results
To perform a holistic view of the results, we used a chemometric multivariate approach. It allows us to examine some independent trends in changes among the investigated properties. They were investigated using hierarchical cluster analysis (HCA, Figure 9) and principal component analysis (PCA, Figure 10).
The analysis was done on a column-wise scaled matrix, as each parameter is ex-pressed with different units. Additionally, two of the parameters, DPPH and ABTS, were expressed as negative values in this matrix. This was done to convert the strong correlation between them and TPC, TPAC, and TFC from negative to positive. This places the loading vectors in the same direction and allows us to see everything visually in more detail.
It can be clearly seen that the parameters form two distinct groups, expressed as two separate groups of loading arrows and two main branches of the dendrogram. The first group contains antioxidant parameters TPC, TPAC, and TFC, together with DPPH and ABTS. The other parameters form the second group.
The increase or decrease of all investigated parameters in opposite directions is responsible for 75% of total variance and is modeled as PC1. PC2 represents an intercorrelated increase or decrease of all investigated parameters and is responsible for 14% of the variability. Further PCs do not contain any interpretable information (results not shown). The cell viability was assessed after 24-h incubation using the MTT assay. Asterisk (*) denotes significantly different data (p < 0.05, unpaired t-test) compared to the control, namely culture medium without substances-0 μg/mL.

Multivariate Analysis of the Results
To perform a holistic view of the results, we used a chemometric multivariate approach. It allows us to examine some independent trends in changes among the investigated properties. They were investigated using hierarchical cluster analysis (HCA, Figure 9) and principal component analysis (PCA, Figure 10).   The analysis was done on a column-wise scaled matrix, as each parameter is expressed with different units. Additionally, two of the parameters, DPPH and ABTS, were expressed as negative values in this matrix. This was done to convert the strong correlation between them and TPC, TPAC, and TFC from negative to positive. This places the loading vectors in the same direction and allows us to see everything visually in more

Conclusions
The management of acne vulgaris should be "multidirectional". Therapeutics should reduce sebum production, as well as follicular hyperkeratinization of the epidermal cells.
For this reason, they should primarily exhibit antioxidant, anti-inflammatory, and antimicrobial activities, without cytotoxic effects.
Our analysis has been designed due to missing data in the available literature, namely considerable gaps in the knowledge considering C. hissaricus and C. hsingshangensis are observed. The unique chemical composition of the representatives of the Rosaceae family makes it possible to obtain a wide range of pharmaceutical, medicinal, and cosmetic products. Thus, Cotoneaster species seem to be promising candidates for further research. Chemical drugs have limitations because of their high toxic activity, as well as their ability to induce adverse side effects. Thus, a growing interest in the use of herbal medicines for the management of acne and other diseases is increasingly recognized.
Among approximately 2500 species belonging to the family Rosaceae, in vitro antiacne research has been carried out only with a small number of them, namely Rosa damascene [65], Rosa multiflora [66], Prunus jamasakura [67], and apple polyphenols [54]. Interestingly, there is a lack of research on other plants from the Rosaceae family for anti-acne activity and the effect of phytochemicals on the skin. Simultaneously, indisputable evidence that phenolic compounds exhibit the desired effects and contribute to alleviate skin disorders can be found in the available literature [51,68]. Furthermore, clindamycin and kaempferol in combination with quercetin possess more beneficial effects than other formulations [69].
In our in vitro research, we characterized composition and evaluated the biological properties of extracts from C. hissaricus and C. hsingshangensis. Thus, we identified the main compounds present in extracts, as well as determined the antioxidant, anti-inflammatory, antimicrobial, and cytotoxic properties of such extracts.
To sum up our results, it seems to be clear that comprehensive and well-designed future research on phenolic compounds from Cotoneaster in greater detail will constitute significant importance in pharmacy and medicine. Among the studied extracts, the diethyl ether fraction of C. hsingshangensis (CHs-2) exhibited great ability to scavenge free radicals and good capacity to inhibit cyclooxygenase-1, cyclooxygenase-2, lipoxygenase, and hyaluronidase. Moreover, it had the most promising power against microaerobic Gram-positive strains, and importantly, it was non-toxic toward normal skin fibroblasts. Taking into account the value of the calculated therapeutic index (>10), it is worth noting that CHs-2 can be subjected to in vivo study and constitutes a promising anti-acne agent.
To the best of our knowledge, there is no reported study on the chemical composition and skin-related properties of the examined species.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/cells11030367/s1, Table S1: Zones of bacterial growth inhibition of the Cotoneaster extracts and fractions.